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Formaldehyde (FA) is an environmental toxin and widely used in building and decorating materials, which is listed as a class 1 carcinogen. Silver ion (Ag+) has a beneficial bactericidal effect, while it can also cause silver poisoning and skin damage. Therefore, developing a quantitative detection technique for formaldehyde and Ag+ is necessary. In this study, a novel difunctional fluorescent probe (DBP) was designed and synthesized based on the natural product dehydroabietic acid, which could selectively detect formaldehyde and Ag+ through "turn-on" and "turn-off" fluorescence response, respectively. The probe DBP performed satisfactory nanomolar-level detection limit to formaldehyde (38 nM) and Ag+ (19 nM) in ethanol/PBS (3/7, v/v, pH = 7.4) solution. Furthermore, DBP could effectively recognize formaldehyde and Ag+ within a short response time (FA: 15 min, Ag+: 100 s) and a wide pH range (5–10). The sensing mechanisms of DBP to FA and Ag+ were well verified by 1H NMR titration, ESI-MS, DFT computation, and Job's plot analysis. What is more, the applications in bioimaging were carried out in MCF-7 cells and living zebrafish. Besides, the probe DBP was successfully applied in detecting FA and Ag+ in actual food and water samples, and the DBP -loaded filter paper was prepared for convenient tracking gaseous FA and Ag+. • A novel fluorescent probe DBP for detecting formaldehyde and Ag+ was synthesized. • DBP exhibits low LOD, short response time and wide pH range towards FA and Ag+. • DBP was utilized in food and water samples, filter paper test, and bioimaging. [ABSTRACT FROM AUTHOR]